Chlorophytum tuberosum Baker belongs to family Liliaceae and is being used in the indigenous systems of medicine as a galactogogue and aphrodisiac. It is being sold in the market under the common name safed musali. The white tuberous roots of this plant are the medicinally useful parts. The tuberous roots of other species of Chlorophytum, Asparagus, Bombax and Orchids are also sometimes called safed musali leading to confusion. In order to ensure correct botanical standardization to remove the controversy, a detailed pharmacognostic study on tuberous roots of Chlorophytum has been carried out in this study .

Chlorophytum tuberosum Baker belongs to family Liliaceae. In India, it is found in rainfed areas. The plant generally grows along the forest margins, grassy slopes and rocky places along valleys (between 1300 and 2800 m). [1] This is an erect plant growing up to a height of 1.5-2 ft with sheathing leaf base acute to acuminate with entire margin. The roots are tuberous with ellipsoid tubers hanging from them, 10-12 cm long and 1-1.9 cm in diameter. [2] The tuberous roots are medicinally important and are known commonly as safed musali. Safed musali is used as an aphrodisiac and galactogogue [3],[4],[5] as well as for its nutritive, health promoting properties and immunoenhancing, hepatoprotective and antioxidants activities. [6],[7],[8],[9],[10] The tubers are also used in fever, leucorrhoea and also as an aphrodisiac. [10]

The species Asparagus, Bombax and Orchids are also known as safed musali in literature. [3],[4] It is therefore important to define specifications that will allow correct identification of the plant that is being sold as "safed musali". In addition there are 17 species of Chlorophytum recorded in India of which 11 species of Chlorophytum are found to be growing in Maharashtra. [11]

We choose Chlorophytum tuberosum for the present investigation as it is being sold widely in the market under the common name safed musali because of its white tuberous roots.

Material and Methods

Collection and identification of plant materials

The plant materials were collected from in and around Pune district of Maharashtra during the rainy season for correct botanical identification. Efforts were made to collect the plants in flowering and fruiting condition for the correct botanical identification. It was identified with the help of Flora of The Presidency of Bombay. [2] Herbarium specimens were prepared and authenticated from Botanical Survey of India, Western Circle, Pune (India). It is housed in Botanical Garden of Botany Department, Pune. The voucher specimen number is PAVICH2/2009. [12]

Microscopic and macroscopic evaluation

Thin (25μ) hand cut sections were taken from the fresh tuberous roots, permanently double-stained and finally mounted in Canada balsam as per the plant microtechniques method of Johansen. [13] The macroscopic evaluation was studied by the method of Trease and Evans [14] and Wallis. [15]

Histochemical study

The thin transverse sections of fresh root were taken (about 25μ). It was treated with respective reagent for the detection and localization of chemicals in the tissues as per the method of Krishnamurthy. [16]

Phytochemical evaluation

Some roots were dried under the shade so as to avoid the decomposition of chemical constituents, powdered in a blender and finally stored in dry air tied containers for phytochemical screening. Ash and percentage extractive content was measured by following the standard pharmacopoeial techniques. [17] Fluorescence analysis was carried out as per Chase and Pratt. [18] Qualitative phytochemical tests were carried out by standard methods of Harborne [19] and Trease and Evans. [14] Quantitative phytochemical analysis were determined for proteins, carbohydrates and saponins by the methods of Lowry et al., [20] Nelson [21] and Obadoni and Ochuko, [22] respectively. The phytochemical screening was also done by the High Performance-Thin Layer Chromatography (HPTLC). HPTLC study was carried out on Linomat 5 for application using Densitometer-TLC Scanner 3 with "WINCATS" software (Camag, Switzerland). These studies were carried out on pre-coated aluminum fluorescent plates (E. Merck). For HPTLC studies, an extract of methanol (25% GR) solvent system was used and after development, plate was scanned at 254 and 366 nm. [23],[24]

The transverse section of the root had a circular outline. The outermost layer is the epidermis consisting of uniseriate trichomes followed by a very large zone of the cortex. The outermost layer of the cortex just below the epidermis consists of cells which are mostly rectangular, appearing longer than wide. The rest of the cortex are rounded to polygonal parenchymatous cells and have no intercellular spaces. The innermost layer of the cortex is a single-layered endodermis. The stellar structure shows that the endodermis is followed by the pericycle layer. The xylem is exarch variety and the phloem is in between the xylem along with the parenchyma. The central region is occupied by large pith mostly polygonal in shape [Figure 3].

The tuber had a total ash content of 12.6%, the acid insoluble ash being 5.6% [Table 3]. The values of percentage extractives were higher in chloroform and lower in benzene solvent [Table 4]. Fluorescence analysis was carried out to check the purity of the drug. The powder drug was observed in visible light as yellowish brown in color. The powder was treated with nitrocellulose, 1 N sodium hydroxide, 1 N sodium hydroxide in nitrocellulose and dried for 30 min. After this it was observed under ultraviolet light and it emits the color as shown in [Table 5].

Qualitative analysis of the root indicated the presence of proteins, reducing and non-reducing sugars, saponins, fats, tannin, glycoside and alkaloids [Table 6]. The quantity of proteins is higher than saponins and carbohydrates [Table 7]. Saponins are the important chemical and justify the use of tubers of this plant and are used as a well-known health tonic, aphrodisiac and galactogogue. [3],[4],[6],[25] In HPTLC study, the methanolic extract is ultrasonic for 15 min and filtered. The filtrate is used as an application for saponins and stegmasteroids. For each application 20 μl, 10 μl and 5 μl extracts were used and loaded on instrument comprising of Linomat 5 for application using Densitometer-TLC Scanner 3 with "WINCATS" software (Camag, Switzerland). These studies were carried out on pre-coated aluminum fluorescent plates (E. Merck). The plates were scanned at 254 and at 366 nm. [23],[24]

The HPTLC analysis showed that the saponins from the C. tuberosum root samples gave light yellow bands in visible light and blue bands after derivatization in fluorescence light. The plates were scanned at 254 and 366 nm. When images were compared with the graph and table values, it showed maximum area 31.38% at 366 nm after derivatization. The table also indicates the starting Rf values and end Rf values [Figure 4];[Graph 1][Additional file 1],[Graph 2][Additional file 2],[Graph 3][Additional file 3];[Table 8],[Table 9],[Table 10].

The plant C. tuberosum showed the correct taxonomy which is helpful for the standardization of drug; the morphological characters and histochemical study with double staining of the root, percentage extractives, fluorescence and ash analysis and the phytochemical screening of the plant. As in case of saponins and stegmasteroids, the peaks are denoted by the Rf values. These investigations will be useful for the correct botanical identification and authentication of the drug. After getting the overall results of C. tuberosum and if data is comparable with the above mentioned species of safed musali, it can be used as a substitute for them.

Acknowledgements

Both the authors would like to express a sincere thank to Head, Department of Botany, University of Pune, for encouragement and necessary laboratory facilities and V.N. Patil is grateful to authorities of Pune University for providing financial support in the form of research stipend.